Employing siRNA to induce silencing of disease-associated genes in a sequence-specific manner has considerable therapeutic promise [1, 2, 7]. Establishing affinity and specificity for a clinical target is a major challenge for conventional therapeutic approaches involving small molecules or proteins. In contrast, the generality of the endogenous RNA interference mechanism makes the silencing of any known disease-associated gene possible [7-10]. The major obstacles to the clinical implementation of siRNA therapeutics are systemic stability, immunogenicity, and intracellular delivery of nucleic acid. Recent advances have elucidated chemical modification strategies to increase nuclease resistance of administered siRNA sequences and reduce immunogenicity, but delivery remains a challenge [11]. Nanoparticle delivery systems such as liposomal formulations have demonstrated considerable in vivo efficacy, but are highly dependent on the usage of large amounts delivery material that compromise safety [2, 7, 14, 17-21]. For example, the current gold standard of efficacious liposomal siRNA delivery, C12-200, still requires a 10-fold excess of material relative to siRNA [19]. One answer to safe clinical delivery is to develop distinct chemical entities that can be attached directly to the nucleic acid and facilitate delivery while maintaining stability and low immunogenicity, thereby eliminating the need for excess delivery material. A number of small molecule and bioconjugate approaches have been attempted with mixed success, such as cholesterol-siRNA, docosamyl-siRNA, aptamers-siRNA, and TAT peptide-siRNA.
Several lipophilic small molecules have been explored as conjugates for siRNA delivery [11-13]. Attachment of cholesterol to the 3′ position of the sense strand utilizing a pyrrolidine linker yielded cholesterol-siRNA conjugates with improved delivery in cultured cells. In animal experiments, cholesterol-siRNA demonstrated not only significant silencing of apoB protein levels, but also improved pharmacokinetic properties. The conjugation of several other bile acids and lipids to siRNA have also demonstrated similar improvements in both cellular uptake and pharmacokinetic properties. These improvements can be attributed to the interaction of these lipophilic moieties with lipoprotein complexes that enhance serum stability and uptake. While attachment of these lipophilic small molecules succeeded in conferring more drug-like properties, the high doses (50 mg/kg body weight) required are toxic and are a major obstacle preventing clinical implementation with these conjugates. Conjugation of siRNA to synthetic polymers bearing hepatocyte-targeting N-acetylglucosamine ligands resulted in more efficacious delivery at 2.5 mg/kg [22]. These “siRNA dynamic polyconjugates” demonstrate that attachment of large synthetic moieties can achieve reasonable dosing with low toxicity. However, this technology relies on targeting ligands for efficacy and is currently limited to hepatocyte delivery.
Cell-penetrating peptides are highly effective delivery agents that have been implemented successfully as delivery vehicles for proteins, antisense oligonucleotides, and peptide-like nucleic acids [8, 23-36]. TAT trans-activator protein (48-60), transportan, and penetratin are popular cell-penetrating peptides that have been evaluated as potential siRNA delivery conjugates [23, 29, 31, 34, 35]. Peptides were conjugated to the 3′ position of the antisense siRNA strand via a reducible disulfide linkage, giving these conjugates the added potential of removal inside the reducing environment of the cytoplasm after delivery. These peptide-siRNA conjugates demonstrated highly efficacious delivery in cultured cells and down-regulated target genes in mouse models. However, the peptides did not improve in vivo stability, with peptide-siRNA conjugates exhibiting clearance rates similar to naked siRNA. In addition, certain cell-penetrating peptides induced inflammatory and immunogenic responses that would be problematic in a therapeutic context [29].
A number of bioconjugates have also been investigated for their ability to enhance siRNA delivery [37-39]. Receptor ligand-mediated delivery was explored by attachment of insulin growth factor 1 (IGF1) peptide to siRNA [37]. While delivery was improved relative to naked siRNA, this conjugate system could not surpass cholesterol-siRNA for efficacy. While aptamers-conjugated siRNA have demonstrated targeting and improved transfection in proof-of-concept studies, they lack systemic stability, are highly prone to nuclease degradation, and may be unable to induce efficient endosomal escape [40, 41]. Antibody-based targeting systems have received attention for their specificity and high systemic stability and have shown some promising results in an implanted rat tumor model [42, 43]. However, these systems still contend with immunogenicity.
A viable siRNA conjugate system that facilitates delivery without compromising stability or immunogenicity has yet to be identified.
Thus, it is an object of this invention to provide siRNA conjugates that efficiently deliver the siRNA to cells with acceptable stability and immunogenicity.
It is also an object of this invention to provide methods of siRNA treatment using siRNA conjugates that efficiently deliver the siRNA to cells with acceptable stability and immunogenicity.
It is also an object of this invention to provide methods of optimizing siRNA delivery through combination of moieties having different chemical and physical properties.
It is also an object of this invention to provide a compositions for effective delivery of nucleic acids, such as siRNA, to cells and tissues.
It is a further objection of this t invention to provide methods of effectively delivering nucleic acids, such as siRNA, to cells and tissues.